Complementary DNA contains the information coding for the synthesis of proteins. The ability to generate complementary DNA (cDNA) libraries is one of the most fundamental procedures in contemporary molecular biology. Research involving the use of cDNA libraries has already led to significant breakthroughs in our understanding of cancer, AIDS and numerous other medical concerns. Consequently, there is a rapidly expanding commercial interest in this procedure because of its enormous current and future potential applicability. For example, a growing number of companies are marketing "ready made" cDNA libraries or kits which simplify the task of preparing a cDNA library.
While the procedures for generating cDNA libraries are being continuously modified and improved, there are serious drawbacks in the current methods that have not been adequately addressed. As a result, cDNA cloning is generally inefficient, making it both cumbersome and most unfortunately very time consuming.
In standard methods currently used for the preparing of cDNA libraries, the mRNA in the cell is isolated by virtue of the presence of a polyadenylated tail present at its 3' end which binds to a resin specific for this structure (oligo dT-chromatography). The purified mRNA is then copied into cDNA using the enzyme reverse transcriptase, which starts at the 3' end of the mRNA and proceeds towards the 5' end. Second strand synthesis is then performed. Linkers are added to the ends of the double stranded cDNA to allow for its packaging into virus or cloning into plasmids. At this stage, it is in a form that can be propagated, the sum of which is termed the cDNA library.
Unfortunately, the major problem with the actual technology is that the majority of the cDNAs present in any given library are not full-length because the reverse transcriptase enzyme in the majority of cases does not make a complete copy of the mRNA. Obviously, this creates serious problems, especially if one takes into account the fact that the efficiency of copying is inversely proportional to the length of the mRNA. This results in the majority of the genetic information in a cDNA library having an overabundance of incomplete pieces.
Hence, an incomplete or non full-length cDNA usually does not have the entire genetic blueprint required to make a functional protein and is therefore of limited scientific value. Usually, investigators must perform many rounds of isolation (screenings) and construct a "full-length" cDNA from the accumulated pieces. Consequently, valuable time and scientific resources are lost. Obviously, the problem becomes even more acute when long cDNAs are sought. Additionally, some fragments of the desired cDNAs might be so underrepresented in the library that it may be impractical to identify and isolate all the required segments.
Furthermore, in cDNA libraries produced by conventional methods, there is dismal under-representation of sequences close to the 5' end of mRNAs since the reverse transcriptase will usually "fall off" before reaching these sequences. This is unfortunate since there is a growing interest in isolating these 5' proximal sequences, in light of recent studies pointing to the importance of such sequences in regulating gene expression.
Another problem concerning cDNA synthesis is the source and quality of the mRNA used. Using present day technology, the mRNA that is used as a source for cDNA synthesis is purified by its 3' end polyadenylated tail. However, some mRNAs do not possess a 3' end but all mRNAs have a 5' cap structure. Consequently, a cDNA library constructed from this source of mRNA would be more representative of the total genetic information present in the cell. In recent years, unsuccessful attempts have been made to develop antibodies directed against the cap structure of mRNA. The problems usually encountered were related to the insufficient affinity of the antibodies for the cap. This major drawback made it impossible to develop isolation protocols for capped mRNAs.
Therefore, it would be highly desirable to develop a method that would increase the ability of scientists to isolate both full-length cDNA clones and capped mRNA.